Understanding the Molecular Basis of Fragile X Syndrome Using Differentiated Mesenchymal Stem Cells

Objectives Fragile X syndrome (FXS) has been known as the most common cause of inherited intellectual disability and autism. This disease results from the loss of fragile X mental retardation protein expression due to the expansion of CGG repeats located on the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Materials & Methods In the present study, the peripheral blood-mesenchymal stem cells (PB-MSCs) of two female full mutation carriers were differentiated into neuronal cells by the suppression of bone morphogenesis pathway signaling. Then, the expression of genes adjacent to CGG repeats expansion, including SLIT and NTRK-like protein 2 (SLITRK2), SLIT and NTRK-like protein 4 (SLITRK4), methyl CpG binding protein 2 (MECP2), and gamma-aminobutyric acid receptor subunit alpha-3 (GABRA3), were evaluated in these cells using SYBR Green real-time polymerase chain reaction. Results The obtained results indicated that the expression of SLITRK2 and SLITRK4 were upregulated and downregulated in the neuron-like cells differentiated from the PB-MSCs of females with FMR1 full mutation, compared to that of the normal females, respectively. Furthermore, the expression of MECP2 and GABRA3 genes were observed to be related to the phenotypic differences observed in the female FMR1 full mutation carriers. Conclusion The observed association of expression of genes located upstream of the FMR1 gene with phenotypic differences in the female carriers could increase the understanding of novel therapeutic targets for patients with mild symptoms of FXS and the patients affected by other FMR1-related disorders.


Introduction
Fragile X syndrome (FXS, OMIM 309550) is the most common cause of inherited mental retardation that results from the expansion of CGG repeats in the 5' untranslated region (5' UTR) of the fragile X mental retardation 1 (FMR1) gene (1). The incidence of FXS has been estimated approximately at 1 in 4,000 males and 1 in 8,000 females (2). Almost 25% of boys and 6% of girls affected by FXS will develop autism spectrum disorders (ASD); however, only 1-2% of ASD patients presented FXS (3)(4). Men affected by FXS usually exhibit moderate mental retardation and often have physical and behavioral characteristics (5). Females with FMR1 gene full mutation showed intelligence quotient scores within the range of normal to less than 70 points (6). Furthermore, failure in short-term memory, executive function, and visual memory and language impairments are very common in individuals with FXS (7-10).
A genetically-engineered mouse model for FXS did not show characteristics similar to methylation and gene silencing of the FMR1 gene in humans (11). Epigenetic mechanisms in humans and mice are different. Therefore, the mice was not suitable models to study FMR1 epigenetic mechanisms. Furthermore, the differences between mouse and human brain structures reflected the challenges in understanding the molecular mechanisms of abnormal brain development and function in FXS patients. Studies indicated that brain development in humans required more time than in mice. The human brain is also very dependent on interneurons and astrocytes. Clear differences were observed in the nervous system of mouse models and patients with FXS. For example, the development of human interneurons occurs over a prolonged period and requires the integration of unique mechanisms to generate numerous interneurons (12)(13)  MSCs could be isolated from peripheral blood by a non-invasive method as previously published (15).
The product of the FMR1 gene, fragile X mental retardation protein (FMRP), plays an important role in the regulation of the translation of the dendritic messenger ribonucleic acid (mRNA) molecules in response to the activation of synapses (16)(17), indicating that the lack of this protein was associated with mental retardation in males with FXS (18). Although the molecular mechanism of FXS has been revealed to be the expansion of CGG repeats located on the 5' UTR of the FMR1 gene and its abnormal methylation, some studies indicated that the hyper-methylation of the FMR1 gene could directly or indirectly affect the expression of some of its downstream genes (19).

Patients
In this study, 20 ml peripheral blood samples were

Methylation Analysis
The methylation pattern of CGG repeats expansion upstream of the FMR1 gene was determined by methylation-specific polymerase chain reaction (MS PCR) followed by capillary electrophoresis. At first, sodium bisulfite modification was performed on genomic deoxyribonucleic acid (DNA) using EpiTect® Bisulfite (Qiagen, UK). Then, the modified DNA was subject to MS PCR using primers specific for methylated (Met PCR) and unmethylated PCR (non-Met PCR). The primers were as previously described (20). The PCR was

Expression Analysis
In this protocol, the bone morphogenesis pathway in MSCs was suppressed through Noggin treatment.

Patient's Characterization
The present study was performed on a female with a normal allele and two females with FMR1 full mutation. The females who were carriers of FMR1 full mutation showed different phenotypes. One of them presented a normal phenotype; nevertheless, the other female showed the symptoms of FXS, including mental retardation, learning disability, autism, attention deficits, hyperactivity, attentional problems, poor eye contact, obsessional interests or behaviors, shyness or social anxiety, and prominent or large ears.

Assessment of FMR1 Triplet Repeat Region
The TP PCR followed by capillary electrophoresis

Author's contribution
Omrani supervised the project; Z. Fazeli carried out the experiments and Wrote the manuscript; SMH.
Ghaderian contributed to the interpretation of the results; H. Najmabadi helped in the collection of samples.